Cloning and Characterization of a Hybridoma Secreting a 4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK)-Specific Monoclonal Antibody and Recombinant F(ab)
Smokeless tobacco products have been associated with increased risks of oro-pharyngeal cancers, due in part to the presence of tobacco-specific nitrosamines (TSNAs) such as 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK). These potent carcinogens are formed during tobacco curing and as a result...
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| Format: | Article |
| Language: | English |
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MDPI AG
2013-03-01
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| Series: | Toxins |
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| Online Access: | http://www.mdpi.com/2072-6651/5/3/568 |
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| author | Lawrence K. Silbart Michael A. Lynes Gregory P. Marusov John Zinckgraf Stewart K. Richardson Debra Rood Daniel I. Zapata Amy R. Howell Tolga Barker Heather Wanczyk |
| author_facet | Lawrence K. Silbart Michael A. Lynes Gregory P. Marusov John Zinckgraf Stewart K. Richardson Debra Rood Daniel I. Zapata Amy R. Howell Tolga Barker Heather Wanczyk |
| author_sort | Lawrence K. Silbart |
| collection | DOAJ |
| description | Smokeless tobacco products have been associated with increased risks of oro-pharyngeal cancers, due in part to the presence of tobacco-specific nitrosamines (TSNAs) such as 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK). These potent carcinogens are formed during tobacco curing and as a result of direct nitrosation reactions that occur in the oral cavity. In the current work we describe the isolation and characterization of a hybridoma secreting a high-affinity, NNK-specific monoclonal antibody. A structurally-related benzoyl derivative was synthesized to facilitate coupling to NNK-carrier proteins, which were characterized for the presence of the N-nitroso group using the Griess reaction, and used to immunize BALB/c mice. Splenocytes from mice bearing NNK-specific antibodies were used to create hybridomas. Out of four, one was selected for subcloning and characterization. Approximately 99% of the monoclonal antibodies from this clone were competitively displaced from plate-bound NNKB conjugates in the presence of free NNK. The affinity of the monoclonal antibody to the NNKB conjugates was Kd = 2.93 nM as determined by surface plasmon resonance. Free nicotine was a poor competitor for the NNKB binding site. The heavy and light chain antibody F(ab) fragments were cloned, sequenced and inserted in tandem into an expression vector, with an FMDV Furin 2A cleavage site between them. Expression in HEK 293 cells revealed a functional F(ab) with similar binding features to that of the parent hybridoma. This study lays the groundwork for synthesizing transgenic tobacco that expresses carcinogen-sequestration properties, thereby rendering it less harmful to consumers. |
| first_indexed | 2024-04-11T21:57:40Z |
| format | Article |
| id | doaj.art-949b583833534852a086a495af76dde4 |
| institution | Directory Open Access Journal |
| issn | 2072-6651 |
| language | English |
| last_indexed | 2024-04-11T21:57:40Z |
| publishDate | 2013-03-01 |
| publisher | MDPI AG |
| record_format | Article |
| series | Toxins |
| spelling | doaj.art-949b583833534852a086a495af76dde42022-12-22T04:01:02ZengMDPI AGToxins2072-66512013-03-015356858910.3390/toxins5030568Cloning and Characterization of a Hybridoma Secreting a 4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK)-Specific Monoclonal Antibody and Recombinant F(ab)Lawrence K. SilbartMichael A. LynesGregory P. MarusovJohn ZinckgrafStewart K. RichardsonDebra RoodDaniel I. ZapataAmy R. HowellTolga BarkerHeather WanczykSmokeless tobacco products have been associated with increased risks of oro-pharyngeal cancers, due in part to the presence of tobacco-specific nitrosamines (TSNAs) such as 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK). These potent carcinogens are formed during tobacco curing and as a result of direct nitrosation reactions that occur in the oral cavity. In the current work we describe the isolation and characterization of a hybridoma secreting a high-affinity, NNK-specific monoclonal antibody. A structurally-related benzoyl derivative was synthesized to facilitate coupling to NNK-carrier proteins, which were characterized for the presence of the N-nitroso group using the Griess reaction, and used to immunize BALB/c mice. Splenocytes from mice bearing NNK-specific antibodies were used to create hybridomas. Out of four, one was selected for subcloning and characterization. Approximately 99% of the monoclonal antibodies from this clone were competitively displaced from plate-bound NNKB conjugates in the presence of free NNK. The affinity of the monoclonal antibody to the NNKB conjugates was Kd = 2.93 nM as determined by surface plasmon resonance. Free nicotine was a poor competitor for the NNKB binding site. The heavy and light chain antibody F(ab) fragments were cloned, sequenced and inserted in tandem into an expression vector, with an FMDV Furin 2A cleavage site between them. Expression in HEK 293 cells revealed a functional F(ab) with similar binding features to that of the parent hybridoma. This study lays the groundwork for synthesizing transgenic tobacco that expresses carcinogen-sequestration properties, thereby rendering it less harmful to consumers.http://www.mdpi.com/2072-6651/5/3/568TSNAsF(ab)NNKmonoclonal antibody2A cleavage |
| spellingShingle | Lawrence K. Silbart Michael A. Lynes Gregory P. Marusov John Zinckgraf Stewart K. Richardson Debra Rood Daniel I. Zapata Amy R. Howell Tolga Barker Heather Wanczyk Cloning and Characterization of a Hybridoma Secreting a 4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK)-Specific Monoclonal Antibody and Recombinant F(ab) Toxins TSNAs F(ab) NNK monoclonal antibody 2A cleavage |
| title | Cloning and Characterization of a Hybridoma Secreting a 4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK)-Specific Monoclonal Antibody and Recombinant F(ab) |
| title_full | Cloning and Characterization of a Hybridoma Secreting a 4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK)-Specific Monoclonal Antibody and Recombinant F(ab) |
| title_fullStr | Cloning and Characterization of a Hybridoma Secreting a 4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK)-Specific Monoclonal Antibody and Recombinant F(ab) |
| title_full_unstemmed | Cloning and Characterization of a Hybridoma Secreting a 4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK)-Specific Monoclonal Antibody and Recombinant F(ab) |
| title_short | Cloning and Characterization of a Hybridoma Secreting a 4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK)-Specific Monoclonal Antibody and Recombinant F(ab) |
| title_sort | cloning and characterization of a hybridoma secreting a 4 methylnitrosamino 1 3 pyridyl 1 butanone nnk specific monoclonal antibody and recombinant f ab |
| topic | TSNAs F(ab) NNK monoclonal antibody 2A cleavage |
| url | http://www.mdpi.com/2072-6651/5/3/568 |
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